PROJECT SUMMARY: One of Candida albicans' most impressive virulence attributes is the ability to propagate as a biofilm when attached to a medical device, such as a venous catheter. This critical factor alone is responsible for the majority of persistent and disseminated infections. As conventional antimicrobials are ineffective for treatment of these life-threatening infections, further understanding of the biofilm lifestyle, including how the cells survive drug therapy and disperse to distant organs is desperately needed. The microbe-derived extracellular matrix, a distinguishing feature of biofilms, has been linked to several roles in biofilm pathogenesis. The proposed investigation capitalizes on our progress during the last funding period that identified the role of a polysaccharide mannan-glucan complex for biofilm resistance and dispersion. We were surprised to find the maturation process occurred in the extracellular space. However, it was unclear how these matrix materials were delivered and assembled. Our preliminary study of the genesis and role of a lipid matrix component provides compelling evidence for vesicle cargo delivery of matrix components critical for drug resistance. This process also appears to regulate biofilm cell dispersion. Our excitement for future investigation of the function of vesicle cargo in biofilm pathogenesis is based upon four unexpected observations. First, we identified vesicles in the extracellular matrix, and these biofilm-derived vesicles carry a distinct cargo that is different from vesicles of free-living (planktonic) cells. Second, we identified vesicle-defective mutants affecting the ESCRT complex; these mutants are impaired in accumulation of matrix and display phenotypes of other matrix-defective mutants (biofilm drug susceptibility and reduced cell dispersion). Third, the cargo of these vesicle mutants differed from vesicles produced by WT strains. Fourth, several cargo mutants display biofilm resistance and dispersion defects, and are rescued by addition of wild-type extracellular vesicles. Our major objectives now are to define the vesicle cargo responsible for the matrix drug resistance, to define the vesicle cargo responsible for cell dispersion, and to discern the genetic pathways that orchestrate this novel process during biofilm pathogenesis.